Using byproducts to produce biofuels, energy and basic chemical compounds is increasingly more needed in the current oil shortage situation. Many compounds that have conventionally been produced from oil can be biotechnologically synthesized today using renewable resources. This is the case of biologically producing 2,3-butanediol.
2,3-butanediol is an organic compound, specifically an alcohol, the molecular formula of which is C4H10O2. There are three isomeric forms thereof: D-(−)-, L-(+)- and meso-, and it is also known as 2,3-butylene-glycol, dimethylene-glycol, dimethylethylene-glycol, and its name according to IUPAC is butane-2,3-diol. Its molecular weight is 90.121 (g mol−1), and it is found in cocoa butter and in the roots of the plant called Ruta graveolens. 
Interest in this compound has increased considerably in recent years due to the large number of industrial applications it has, primarily in the chemical and energy industry. Both 2,3-butanediol and some of its derivatives are used in the production of plastics and solvents. Given its high octane rating, 2,3-butanediol is useful as an octane enhancer in fuels. Given its low melting point (−60° C.), it is also used as antifreeze. As an analytical reagent, 2,3-butanediol is used for resolving carbonyl compounds in gas chromatography.
One of the main applications of 2,3-butanediol is its conversion into 1,3-butadiene, which is used for synthetic rubber production. In addition, the 2,3-butanediol dehydrogenation product, diacetyl, is a highly valued bacteriostatic flavoring agent in the food industry. 2,3-butanediol dehydration yields methyl ethyl ketone (MEK), which is an additive with high combustion heat used in fuels. MEK is also used as a resin and lacquer solvent. Polyurethane-melamides (PUMAs), which are useful in cardiovascular applications, are obtained from 2,3-butanediol esterification with malic acid. Other 2,3-butanediol esterification products are used in cosmetics and in the pharmaceutical industry. Finally, the production of wetting agents, elastane, fumigants, plasticizers (such as polyvinyl chloride, cellulose nitrate and polyacrylates, for example), perfumes, printing inks, softeners and drug vectors are also considered potential applications of 2,3-butanediol.
Various microorganisms are capable of accumulating 2,3-butanediol, such as, for example, strains of the bacterial species Aeromonas hydrophila, Aerobacter indologenes, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus polymyxa, Bacillus subtilis, Brevibacillus brevis S1, Corynebacterium glutamicum, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella terrigena, Lactobacillus brevis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus plantarum, Lactococcus lactis, Lactococcus lactis subsp. lactis var. diacetylactis, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Oenococcus oeni, Pediococcus pentosaceus, Pseudomonas chlororaphis, Raoultella terrigena, Serratia marcescens, Streptococcus faecalis, some rhizobacteria and marine algae Chlamydomonas perigranulata, although not all of them do so in significant amounts. Some yeasts are also capable of synthesizing 2,3-butanediol, but with very low productivity, so the only microorganisms of industrial importance for production of this compound are bacteria.
It is furthermore known that 2,3-butanediol synthesis seems to play a very important physiological role for microorganisms as it prevents acidification, regulates the NADH/NAD+ ratio and stores carbon and energy for growth. The genes responsible for 2,3-butanediol conversion from A. aerogenes and K. terrigena have been cloned and characterized. They are an operon called budABC consisting of three genes encoding three key enzymes: α-acetolactate synthase, α-acetolactate decarboxylase and acetoin reductase (2,3-butanediol dehydrogenase).
The bacteria identified up until now as the most efficient among 2,3-butanediol producers are B. polymyxa, K. oxytoca and K. pneumoniae, and they primarily use sugars as a substrate.
In addition, it is well known that the ideal solution in base chemical compound production is the bioconversion of industrial byproducts (such as glycerol, whey or agricultural waste) or the use of excess biomass (such as wood hydrolysate). Particularly, and given that about 100 Kg of crude glycerol are generated per ton of product in biodiesel production, and taking into account the increase in biodiesel production, finding alternatives for later use of this byproduct is of interest. For this reason, glycerol is known as one of the potentially most interesting substrates for producing 2,3-butanediol.
In the fermentation processes for converting glycerol into products other than 2,3-butanediol, such as 1,3-propanediol, the participation of several strains belonging to the species K. pneumoniae, for example, the K. pneumoniae strain DSM 2026, which is a very good glycerol fermenter, stands out. In turn, K. pneumoniae strain G31 is better under fermentation conditions in which the pH value is not controlled.
These species are also capable of producing 2,3-butanediol from glycerol. In batch feed fermentation processes, K. pneumoniae G31 produced 2,3-butanediol with a yield of 0.36 g/g (Petrov & Petrova, 2009)6, and in assays with forced pH variations, the yield was 0.39 g/g, produced from glycerol in both cases (Petrov & Petrova, 2010)7. However, species K. pneumoniae is classified as a group 2 biological agent, which means that it is a pathogenic agent that can cause diseases in humans and be a risk for workers (Directive 2000/54/CE of the European Parliament and of the Council of 18 Sep. 2000), so strains of this species are not suitable for use in industrial biotechnology.
With respect to patent documents, patent document U.S. Pat. No. 5,254,467 relates to a process for the conversion of glycerol into 1,3-propanediol by means of microorganisms. Said process comprises fermenting said microorganisms in a medium containing 5-20% by weight of glycerol under standard anaerobic fermentation conditions, subsequently recovering the produced 1,3-propanediol, and 2,3-butanediol as a secondary product. The patent document mentions Klebsiella planticola strain IAM 33 as a possible microorganism useful for these purposes. Nevertheless, the 2,3-butanediol production yield in this fermentation process is very low so use thereof is not feasible on an industrial level.
Patent document JP 2010226959 discloses the use of other microorganisms with the ability to generate ethanol from glycerol, specifically Raoultella ornithinolytica and R. planticola, even though it does not mention producing 2,3-butanediol.
The object of patent document US2007/0148749 A1 is to improve 1,3-propanediol production from glycerol using certain bacteria strains belonging to genera Caloramator, Citrobacter, Clostridium, Enterobacter, Escherichia, Klebsiella, Lactobacillus, Listeria and Salmonella using a reaction catalyzed by enzymes in which the first step is converting glycerol into 3-hydroxypropionaldehyde and water, and the second step is reducing 3-hydroxypropionaldehyde to 1,3-propanediol. It does not mention producing 2,3-butanediol.
The object of the invention of patent document US 2008/0274522 A1 is a method for the 2-butanone production by means of fermentation by a microorganism. The method uses the enzyme acetolactate synthase and temperature reduction during the fermentation process, resulting in higher tolerance of the host to butanone. It does not mention producing 2,3-butanediol.
Patent application WO 2007/130518 A2 relates to 2-butanol production by means of the industrial fermentation of a recombinant microorganism. The transgenic host contains at least one recombinant DNA molecule containing a gene encoding a polypeptide with the ability to catalyze a substrate and to perform conversion of: i) pyruvate into alpha-acetolactate; ii) alpha-acetolactate into acetoin; and iii) acetoin into 3-amino-2-butanol. It does not mention producing 2,3-butanediol.
Patent document EP 1892300 A1 provides a method for producing 1,3-propanediol starting from crude glycerol, a byproduct obtained during biodiesel production, and using as fermentation microorganisms Clostridium butyricum, Clostridium pasteurianum and Klebsiella pneumoniae. It does not mention producing 2,3-butanediol.
All the other significant examples of producing 2,3-butanediol refer to the use of sugars (e.g. glucose) as a substrate.
Therefore, the technical problem of the present invention relates to providing novel strains of the species R. planticola, that can be used in the industrial production of 2,3-butanediol from glycerol. Said technical problem is preferably resolved by providing two novel strains of R. planticola designated in the present description as IA1 and IIIA3 and deposited on Dec. 6, 2012 with accession number CECT8158 (corresponding to IA1) and accession number CECT8159 (corresponding to IIIA3) in the Spanish Type Culture Collection (CECT), Parc Cientific Universitat of Valencia, c/Catedrático Agustín Escardino, 9, 46980 Paterna—Valencia, Spain, according to the provisions of the Budapest Treaty. Said strains have a 2,3-butanediol production capacity that is better than other strains in the same species, so the present invention also contemplates an industrially viable method for the biotechnological conversion of glycerol into 2,3-butanediol using said novel strains or mutants thereof.